Background and Aim: The affinity of an antibody with its antigen is a crucial parameter in its biological activity and carrying out immunologic tests such as radioimmunoassay (RIA) and immunohistochemistry. This study was done in our laboratory at Tarbiat Modarres University to measure the affinity constant of specific monoclonal antibodies/MAB (A1G8F7) with alkaline phosphatase enzyme produced.Material and Methods: Several Methods have been innovated to determine the affinity and avidity of antibodies. In all of them a serum is used in which antigen and antibody would reach balance and, without a change in the balance, free antigen and antigen associated with antibody are measured. Applying a rapid and simple ELISA-based method the affinity constant of specific MAB (A1G8F7) against alkaline phosphatase was measured.Results: Constant density of antibody for use in specific ELISA used to determine dissociation constant (Kd), applying klotz methodology-by means of a competitive ELISA proved to be 1μg Kd of A1G8F7 clone; through klotz fig, it was estimated to be 3.8×10-9Conclusion: considering that the Kd of natural antibodies in the body for most antigens ranges from 10-7 to 10-11 and that the less Kd the more affinity of the antibody, the antibody secreted by A1G8F7 clone seems suitable. Practically, in immunocytochemistry for most immunologic measuring, histochemistry test done with mAb-ALP enzyme in three line-staining led to pretty good and comparable results with the similar trade sample of APAAP (Dako, Denmark).